In this Phase I STTR proposal, our R&D goal is the development and evaluation of a novel functionally-graded biohybrid vascular graft for small diameter (<6 mm) coronary bypass applications. Tissue engineered vascular constructs developed to date have mostly utilized synthetic and animal- derived biomaterials and require pre-seeding of host cells before implantation to overcome the complications of prosthetic vascular grafts. However, these graft systems exhibit many limitations including poor cellular adhesion, inadequate biomechanical and functional properties. The PI has recently demonstrated an in vitro regenerated human endothelium on functionally-layered polymeric scaffolds containing bioactive proteins. Moreover, VBI has developed a unique human biomatrix (HuBiogel) that allows viable tissue constructs by cultivating single or multiple cell types. HuBiogel milieu can also be controlled via enrichment with specific growth factors (VEGF). We now propose to combine our functionally-layered graft strategy with this physiological HuBiogel technology for fabricating an advanced 3D vascular construct demonstrating enhanced lumen endothelialization and biocompatibility and structural integrity. Phase I specific aims are: 1) Fabricate and optimize functionally layered HuBiogel-biohybrid scaffolds (4 mm ID) with promising biomechanical properties using our sequentially co-spun HuBiogel/polymer nanomatrix formulation protocol; and 2) Evaluate in vitro anti-thrombogenicity, biocompatibility and functionality of new HuBiogel-hybrid graft with human vascular endothelial and skeletal muscle cells using established bioreactor culture methods. In addition, a pilot animal study will be performed to demonstrate in vivo biomechanical integrity of new vascular graft by implanting in rabbit aorto-iliac model. We anticipate that new biohybrid device engineered with bioactive HuBiogel (functional endothelium) and polymeric gradient (robust biomechanics) will provide an improved ready-to-implant for small diameter grafting without requiring pre-seeding of primary cells. Thus, successful development of prototypic biohybrid vascular graft will form important basis for detailed quality control analyses and in vivo animal validation studies (dog or pig coronary bypass models) in future Phase II. For this STTR proposal, a dedicated team of bioengineers, cell biologist and cardiologists is gathered to develop and commercialize a novel small- diameter human vascular graft for coronary replacement. Potential for technological innovation and commercial application: No biohybrid vascular graft employing human biomatrix scaffold design is currently in market. We anticipate that a ready-to- implant or graft device will have worldwide market for the bypass treatments, estimated to be in millions of dollar.